Phorbol 12-myristate 13-acetate (PMA) potently induces apoptosis of LNCaP human prostate cancer cells. Here, we show that C4-2 cells, androgen-hypersensitive derivatives of LNCaP cells, also are sensitive to PMA-induced apoptosis. Previous reports have implicated activation of protein kinase C (PKC) isozymes alpha and delta in PMA-induced LNCaP apoptosis using overexpression, pharmacological inhibitors, and dominant-negative constructs, but have left unresolved if other isozymes are involved, if there are separate requirements for individual PKC isozymes, or if there is redundancy. We have resolved these questions in C4-2 cells using stable expression of short hairpin RNAs to knock down expression of specific PKC isozymes individually and in pairs. Partial knockdown of PKCdelta inhibited PMA-induced C4-2 cell death almost completely, whereas near-complete knockdown of PKCalpha had no effect. Knockdown of PKCepsilon alone had no effect, but simultaneous knockdown of both PKCalpha and PKCepsilon in C4-2 cells that continued to express normal levels of PKCdelta inhibited PMA-induced apoptosis. Thus, our data indicate that there is an absolute requirement for PKCdelta in PMA-induced C4-2 apoptosis but that the functions of PKCalpha and PKCepsilon in apoptosis induction are redundant, such that either one (but not both) is required. Investigation of PMA-induced events required for LNCaP and C4-2 apoptosis revealed that p38 activation is dependent on PKCdelta, whereas induction of retinoblastoma protein hypophosphorylation requires both PKC signaling pathways and is downstream of p38 activation in the PKCdelta pathway.